【作者】 古莹；

【导师】 徐荣臻；

【作者基本信息】 浙江大学 ， 内科学， 2007， 硕士

【摘要】 白血病是一类常见的严重威胁人类健康的造血系统恶性肿瘤。目前，化疗和造血干细胞移植是治疗白血病的主要手段，但是，通过化疗只能取得短暂缓解，而异基因造血干细胞移植治疗白血病5年无病生存率仅为35％—50％。此外，因造血干细胞供体限制，只有部分患者能进行这一治疗，而且还存在移植相关死亡和复发危险性。因此，寻找新的更有效抗白血病药物仍然十分迫切和需要。氯原酸是一种提取自中草药的物质，具有广泛的生物学效应，氯原酸钠是一种氯原酸和钠盐的半合成小分子化合物，近期有研究显示氯原酸可以诱导包括Bcr-Abl阳性的慢性粒细胞白血病（CML）原代细胞在内的Bcr-Abl阳性细胞的凋亡，而对Bcr-Abl阴性的细胞无明显作用。另外，我们前期的研究工作已经证实，钙离子通道阻止剂小檗胺能选择性诱导Bcr-Abl阳性的人白血病细胞系K562和原代白血病细胞凋亡，而对正常骨髓造血细胞毒性很少。基于此，我们采用人白血病细胞系KG1、K562和Jurkat，以适当浓度的氯原酸作用于各细胞系，观察氯原酸对各株白血病细胞的增殖抑制作用。并将氯原酸和小檗胺联合作用于K562细胞，比较药物联合作用和各自单独作用对细胞的增殖抑制作用。对象与方法一、研究对象人白血病细胞系：KG1、K562和Jurkat三株细胞系，其中KG1为急性原始粒细胞白血病细胞系，K562为慢性粒细胞白血病细胞系，Jurkat为急性淋巴细胞白血病细胞系（ALL）。均为本实验室常规培养。二、研究方法1．细胞接种：采用处于对数生长期的细胞，按细胞密度8×10³／孔分别加入96孔细胞培养板中。2．药物作用：每孔分别加入梯度浓度的药物，Na-氯原酸和（或）小檗胺，不加药物为阴性对照，每组设3个平行孔。37℃，5％CO₂，饱和湿度下培养48小时。3．MTT作用：每孔加入20μlMTT，继续培养4小时。4．测吸光度：离心，弃上清，每孔加入200μl二甲基亚枫，水平摇床振荡5分钟，用全自动酶标光度计在470nm处测吸光度。5．在计算机中使用Microsoft Office Excel计算抑制率及50％细胞生长抑制浓度，并以药物浓度为横坐标，活细胞数为纵坐标作图，观察药物对细胞增殖抑制的情况。结果-20℃保存的不同浓度的氯原酸钠对K562细胞有明显的增殖抑制作用，其作用强度呈现浓度依赖性，48小时的50％细胞生长抑制浓度（IC50）值为24.210μg／ml，但是对KG1、Jurkat均无明显得增殖抑制作用。4℃保存的不同浓度的氯原酸钠对K562细胞具有更强的增殖抑制作用，48小时的IC50值为13.177μg／ml，同时对Jurkat细胞也有较明显的增殖抑制作用，48小时的IC50值为13.177μg／ml，作用强度呈现浓度依赖性，但对KG1细胞仍无作用。小檗胺单独作用对K562细胞有明显的增殖抑制作用，其作用强度呈现浓度依赖性。48小时的IC50值为6.080μg／ml。13μg／ml（IC50）的氯原酸钠与不同浓度的小檗胺联合作用对K562细胞的增殖抑制作用略强于小檗胺单独作用于K562细胞。6μg／ml（IC50）的小檗胺与不同浓度的氯原酸钠联合作用对K562细胞的增殖抑制作用明显强于氯原酸钠单独作用于K562细胞。结论1．-20℃避光保存的氯原酸钠可以选择性抑制bcr-abl阳性的CML细胞系K562细胞的增殖。2．4℃保存的氯原酸钠对bcr-abl阳性的CML细胞系K562的增殖抑制作用增强，并且对bcr-abl阴性的Jurkat细胞亦具有增殖抑制作用。3．氯原酸钠与小檗胺具有协同抑制CML白血病细胞K562增殖的作用。更多还原

【Abstract】 Leukemia is the most common malignant neoplasm of hematopoietic system. nowadays,chemical therapy and haemopoietic stem cell transplantation are main measures of leukemia therapy.But , chemical therapy can only gain transient relief and 5-year disease free survival rate of haemopoietic stem cell transplantation is 35% — 50 % .In addition,because of the constraint of stem cell donor,only a few people can receive HSCT . HSCT exsits many chancinesses associated with transplantation and recurrence. For the past many years, hematologists have been searching for medicines to cure leukemia worldwild. In a recent study, a Bcr-Abl kinase inhibitor was identified from an herbal source. The compound was characterized to be chlorogenic acid （Chl）. Chlorogenic acid has diverse biologic activities. Chl inhibits Bcr-Abl kinases and induces apoptosis of Bcr-Abl-positive cells including Bcr-Abl-positive primary leukemic cells of CML patients in vitro. Berbamine is a calcium ion channel blocker. In our past study, berbamine exhibits selective growth inhibitory effects on Bcr-Abl-positive CML cell line K562 including Bcr-Abl-positive primary leukemic cells of CML patients in vitro and have little cytotoxicityto normal myeloid element. Accordingly, leukemic cell lines of KG1 、 K562 and Jurkat are adopted and affected debita spissitudine chlorogenic acid. We observe the inhibition of cells treated by Chl. We also observe the unification of Chl and berbamine.Materials and methodsMaterialsHuman leukemia cell lines:KGl,K562 and Jurkat cell lines. KGl cell line for AML-M1, K562 cell line for AML-M6, and Jurkat for acute lymphoblastic leukemia （ALL）.Methods1. Cell inoculation: We use cell lines on logarithmic growth phase. Inoculum density is 8×10³/well.2. Drug treatment: Na-chlorogenic acid and berbamine treat cell lines on graded concentration. Blank well is negative control. Cells are cultivated in 37°C, 5% CO₂ incubator for 48 hours.3. MTT effect: Adding 200ulMTT per well and cultivating for 4 hours.4. Measuring absorbance: centrifuging and discarding supernatant. Adding 200ul DMSO per well. Agitating for 5 minutes and measuring absorbance at 470nm.5. Calculating inhibition ratio and IC50 .Plotting to observe apoptosis.ResultsNa-chlorogenic acid in —20°C exhibits growth inhibitory effects on Bcr-Abl-positive CML cell line K562 in concentration dependent. IC50 is 24.210ug/ml,but have no inhibitoryeffects on KG1,Jurkat cell lines. Na-chlorogenic acid in 4°C exhibits stronger growth inhibitory effects on K562,and have inhibitory effects on Jurkat. It has no growth inhibitory effects on KG1. IC50（K562） is 13.177 ug/ml, IC50（Jurkat） is 19.640ug/ml.Berbamine induces apoptosis of Bcr-Abl-positive cell line K562 in concentration dependent. IC50 is 6.080ug/ml.The unification of Na-chlorogenic acid and berbamine on K562 is stronger than solo effect of Na-chlorogenic or berbamine.Conclusions1. Na-chlorogenic acid in — 20°C exhibits selective growth inhibitory effects on Bcr-Abl-positive CML cell line K562.2. Na-chlorogenic acid in 4°C exhibits stronger growth inhibitory effects on K562,and have inhibitory effects on Jurkat.3. Chlorogenic acid and berbamine have co-inhibitory effect on K562.更多还原